This proposal deals with the application of nmr to study the unfolding and refolding of bovine pancreatic ribonuclease (RNase) and derivatives in an attempt to detect intermediate states. The behavior of many individual amino acid residues will be following during the unfolding transition. For certain amino acids, this will require chemical modification of the enzyme. NMR unfolding experiments on the proteolytic derivatives of RNase (RNase-S and RNase-P) should shed light on the roles of the N- and c-termini in stabilizing the native structure. These experiments should provide information as to the roles played by the various segments of the enzyme in maintaining the native three dimensional structure and also provide information about the pathway by which the enzyme folds into this unique structure. In addition, this proposal seeks to characterize some unusual derivatives of RNase. These products have 2-4 times the activity of native RNase under standard assay conditions. Our working hypothesis is that they represent RNase-mercaptoethanol mixed disulfides.